Genomic Surveillance of Listeria monocytogenes in Taiwan, 2014 to 2019

ABSTRACT Listeria monocytogenes is a life-threatening foodborne pathogen. Here, we report the genomic characterization of a nationwide dataset of 411 clinical and 82 food isolates collected in Taiwan between 2014 and 2019. The observed incidence of listeriosis increased from 0.83 to 7 cases per million population upon implementation of mandatory notification in 2018. Pregnancy-associated cases accounted for 2.8% of human listeriosis and all-cause 7-day mortality was of 11.9% in nonmaternal-neonatal listeriosis. L. monocytogenes was isolated from 90% of raw pork and 34% of chicken products collected in supermarkets. Sublineages SL87, SL5, and SL378 accounted for the majority (65%) of clinical cases. SL87 and SL378 were also predominant (57%) in food products. Five cgMLST clusters accounted for 57% clinical cases, suggesting unnoticed outbreaks spanning up to 6 years. Mandatory notification allowed identifying the magnitude of listeriosis in Taiwan. Continuous real-time genomic surveillance will allow reducing contaminating sources and disease burden. IMPORTANCE Understanding the phylogenetic relationship between clinical and food isolates is important to identify the transmission routes of foodborne diseases. Here, we performed a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes. We show a 9-fold increase in listeriosis reporting upon implementation of mandatory notification. We found that sublineages SL87 and SL378 predominated among both clinical (50%) and food (57%) isolates, and identified five cgMLST clusters accounting for 57% of clinical cases, suggestive of potential protracted sources of contamination over up to 6 years in Taiwan. These findings highlight that mandatory declaration is critical in identifying the burden of listeriosis, and the importance of genome sequencing for a detailed characterization of the pathogenic L. monocytogenes genotypes circulating in Asia.


Introduction:
Line 60: delete "(Central nervous system (CNS) infection)" Line 72: change "e.g." to "e.g.," throughout manuscript Line 75: change "16) and showing higher" to "16). These exhibit higher..." Methods: Line 96: why were raw chicken, raw ground pork, and vegetables chosen? Specifically, raw chicken and raw ground pork would not be consumed raw? As well, need to be specific with respect to vegetables -which ones, condition, etc. As well, how representative is this choice of commodities? Line 101: why 100 ml? All culture methods usually have a 1:10 start? Line 119: what is "--only-assembler and --careful options" mean?
Results: Line 144: "327 human listeriosis cases" -on line 483 it lists 411? Line 165: as previous comment, please identify what vegetables were chosen and provide data (are they raw, transformed, etc.) Line 185: "suggesting unnoticed long-term outbreaks." -is there any epidemiological evidence to support this? Otherwise, suggest to tone down the sentence as it may be a common CT may be prevalent? Discussion: Line 246: "of circulating clones" -how would this be tested? Line 246: with respect to low fertility rates, is this actually the inability of women to conceive? Or that people are not having children? As well, can you not average/normalize the numbers/cases to get a better indication of the comparison to Europe and North America Line 271: Not sure that it is suggestive of long-term contamination as there is no retail data/survey or any epidemiological evidence to support that claim. Remove and/or re-write.
General comments: -try not to start sentences with numbers - Table 1 (and elsewhere) -is it easier to speak about CCs (authors talk about SLs, etc. which may be less common vernacular - Table 2  Authors describe their results as "a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes." In my opinion, if this (nationwide study) is true for clinical cases of listeriosis, it is not true for food. As far as I understood, foods sampled are not high risk-foods for listeriosis. Please provide further information if I'm not correct.
Line 34: I think it was not the "observed" but the "reported" Line 63: "0.1 and 12 per million people" Where? Line 64: Please provide the latest reports (3, 4). Line 65 and elsewhere in the manuscript: numbers less than ten should be written out in full Line 71 and elsewhere in the manuscript: Avoid abbreviations at the beginning of a sentence Lines 91-93: Is Taiwan Centers for Disease Control the only intuition receiving information/ isolates from listeriosis cases in the country? Please provide information Lines 93-95: Do you mean ethical approval for this study? Lines 95-96: Please provide more detailed information for: "To assist epidemiological investigations, food samples (raw chicken, raw ground pork and vegetables)...". As listeriosis is mainly associated with the consumption of ready-to-eat products and is destroyed during cooking, please explain how these results could "assist epidemiological investigations" Line 102 and elsewhere in the manuscript: insert space between "37" and "{degree sign}C" Lines 102-106 and elsewhere in the manuscript: Please avoid long sentences. Rephrase "Then 0.1 ml of the overnight culture was transferred to 10 ml of Listeria enrichment Fraser broth and incubated at 37{degree sign}C, 200 rpm for 18 to 24 hr. The 10 μL of secondary enrichment broth was seeded onto Chromogenic Listeria Agar (CMP, Taiwan) and incubated at 37{degree sign}C for up to 48 h, and the suspected colonies were purified in Brain Heart Infusion Agar (CMP, Taiwan) cultured at 37{degree sign}C for 18 to 24 h. Lines 134-135: Only found 445 sequences. Please check. 165-166: "By contrast, all the vegetable products sampled in 2019 (n=50) were negative for L. monocytogenes. " What type of products? Lines 181: Are the sample products eaten raw? Lines 185: or widely distributed CTs? Line 231: in my opinion, we can not claim "epidemiological ... study" Lines 235-242-"As a result, since July 2021, the Taiwan Food and Drug Administration (TFDA) has extended food monitoring of Listeria from infant and dairy products to refrigerated and ready-to-eat food products (https://consumer.fda.gov.tw). As compared to the regulation before this date where food products were randomly sampled once, an extended sampling plan (5 to 10 sampling times per location according to the guidelines on sampling CAC GL 50-2004) is now in place for food microorganisms. This may help to identify more contamination sources of L. monocytogenes in different food products." Useful information but probably not in this context" Lines 243-245 "As compared to Europe and North America ...." this should be supported with references from official entities. Fig 3-The figure looks very nice, but it is difficult to read. A supplementary table with all the information for each isolate would be useful.

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Point-by-point responses
We appreciated the valuable comments from editor and reviewers. We answered the questions/concerns in the following text, made several modifications and rephrased paragraph especially on the details of food sampling. We provided a table describing food sampling details (Table S1), and a table that links to Figure 3, summarizing genomic characteristics of the 493 isolates included in this study (Table S2). We hope we have addressed all the points and sincerely thank for your interest of our work.
Editor comments: In addition to the reviewer comments below, please provide more details on the food sampling methodology (rationale for selection, vegetable types, was each commodity sampled every two weeks, how many markets per city, per sampling time? were the sampling locations kept constant throughout the study period?) We focused on chicken and pork products as these food commodities are the two main meat types consumed by Taiwanese, and are mostly produced locally in Taiwan. Foods made by these meat types are at high risk of being undercooked in some Chinese cuisine styles, thus a likely source of contamination. We included now the detailed sampling information in the revised manuscript (L93-101 and the new Table S1). Thank you.
L96 raw ground pork, and vegetables (add the comma) We followed this suggestion (L94). Thank you.
In the text can tie your findings together, for example, were any linkages or trends were noted in virulence, stress, AMR gene presence and clinical/food isolation source.
Similar to other studies, virulence and resistance traits found in this study were more linked to the phylogeny of the isolate rather than the source type. Only comK truncations were significantly associated with food isolation source. This information is now detailed in the revised manuscript (L233-L235). Thank you.
The manuscript states 493 strains were sequenced. The Bioproject related to this manuscript contains 422 entries in the BioSample, and 445 in the SRA. Please correct this discrepancy.
The 445 SRA are those open for public in the Bioproject, and not all the sequences included in this study are publicly available now. Nevertheless, all sequences concerning the 493 isolates in this study have been deposited in SRA (Bioproject PRJNA493675) and will be made publicly available upon acceptation. Isolate accession numbers are provided in the new Table S2).

Reviewer comments:
Reviewer #1 (Comments for the Author): Introduction: Line 60: delete "(Central nervous system (CNS) infection)" We followed the suggestion and deleted this in the revised manuscript (L60).

Methods:
Line 96: why were raw chicken, raw ground pork, and vegetables chosen? Specifically, raw chicken and raw ground pork would not be consumed raw? As well, need to be specific with respect to vegetables -which ones, condition, etc. As well, how representative is this choice of commodities?
We focused on chicken and pork products as these food commodities are the two main meat types consumed by Taiwanese, and are mostly produced locally in Taiwan. We began with raw meat products as in some Chinese cuisine styles these two meat products may be undercooked and possibly leading to cross contamination to cooked food during preparation. The 50 vegetable products are ready-to-eat mixed salad products sold in supermarkets. We aimed to have the samples representing the whole city and thus sampled 46 markets of 5 districts in Taichung, 16 markets of 4 districts in Taipei, and 16 markets of 3 districts in New Taipei cities. We included the sampling information in the revised manuscript (L93-101).
Line 101: why 100 ml? All culture methods usually have a 1:10 start?
We followed the guidance of the Taiwan FDA. As the starting food samples are not in liquid, and 25 g of meat is not easy to be homogenized. Incubation with 100 ml broth would allow complete soaking of the food samples according to our experience.
These options refer to the assembler parameters for high-accuracy assembly. This is now clarified in the text (L123-125).
We have 411 human isolates in this study, of which 78 isolates were collected in 2014-2017, and 333 isolates were collected in 2018-2019 upon mandatory notification.
Among the 333 isolates in 2018-2019, disease information was available from 327 human listeriosis cases. This is now made clear in the text (L143-148).
Line 165: as previous comment, please identify what vegetables were chosen and provide data (are they raw, transformed, etc.) The vegetables are ready-to-eat mixed salad products sold in supermarkets. We included this information in the revised manuscript (L94-95 and the new Table S1).
Line 185: "suggesting unnoticed long-term outbreaks." -is there any epidemiological evidence to support this? Otherwise, suggest to tone down the sentence as it may be a common CT may be prevalent?
Several studies have shown that isolates belonging to the same cgMLST type (CT) are likely to share an epidemiological link (eg., Moura et al., Nat. Microbiol., 2016;Schjørring et al., Euro Surveill., 2017;Van Walle et al., Euro Surveill., 2018). However, in this study we do not have trace-back trace-forward investigations to confirm the source of the outbreaks. We thus follow the suggestion and rephrased this to "suggesting high prevalence of common CTs for several years, which may imply unnoticed long- Line 246: with respect to low fertility rates, is this actually the inability of women to conceive? Or that people are not having children? As well, can you not average/normalize the numbers/cases to get a better indication of the comparison to Europe and North America According to the public information from the Central Intelligence Agency of the United States of the America, the total fertility rate is 1.84 children born/woman in the U.S.A. and 2.03 children born/woman in France, while it is 1.08 born/woman in Taiwan.
However, it is difficult to do normalization with statistical power to test the difference as we may need additional information, such as the detail of total fertility rate calculation that we do not have for analysis. We also agree that the low fertility rate in Taiwan could be due to the inability of women to conceive or that people are not having children, both are correlated with each other.
Line 271: Not sure that it is suggestive of long-term contamination as there is no retail data/survey or any epidemiological evidence to support that claim. Remove and/or rewrite.
We agree that analysis of isolates from food processing and retailing environments is required to make the conclusion. We thus rewrote the sentence to be clearer (L277-280).
General comments: -try not to start sentences with numbers We followed the comment and revised the manuscript accordingly. Thank you.
- which may be less common vernacular In this study we employed cgMLST, which provides a high resolution of circulating strains in comparison to MLST. It is thus more precise to refer to SLs, which are assigned by cgMLST analysis, instead of CCs that are based on 7-gene MLST. To address the reviewer's comment and to be clearer for readers, we now include both nomenclatures in Tables and throughout the text when relevant.
- Table 2 -hard to see the gray boxes? Consider bolding?
We now highlight the largest CTs in bold to be clearer to readers. Thank you.
- Figure 2 -of the 493 isolates, are any duplicates from the same sample? Authors should mention this. As well, are there samples where there was actual diversity (i.e., more than one CC, etc?) Multiple isolates belonging to the same CT from one sample were seen as duplicates, and we only took one from the duplicates for epidemiological analyses. We included this information in the revised manuscript (L111-113).
We have observed strain diversity in two raw ground pork products sampled in Taichung. This information is now also included in the text (L188-190).

Reviewer #2 (Comments for the Author):
Authors describe their results as "a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes." In my opinion, if this (nationwide study) is true for clinical cases of listeriosis, it is not true for food. As far as I understood, foods sampled are not high risk-foods for listeriosis.
Please provide further information if I'm not correct.
While ready-to-eat food products are the main high-risk foods for listeriosis outbreak in the literature, we could not exclude the involvement of undercooked foods, which are the main high-risk foods for infection of Campylobacter and Salmonella, in sporadic listeriosis. No cluster infection of L. monocytogenes at a specific place in a short period has been reported in Taiwan. We thus speculated that inappropriate food processing, such as ingestion of undercooked meat, may account for the sporadic listeriosis cases.
Indeed, L. monocytogenes can be isolated from raw meats (Luo et al., Food Control, 2017;Wang et al., Front Med., 2018). We thus decided to begin with sampling of raw meat products in Taiwan.
Line 34: I think it was not the "observed" but the "reported" The incidence shown here has not been reported and is one of the findings in this study.
These incidences were reported in Europe and the U.S.A.. We updated the references to those published in two years. Please see the references 3-5 in the revised manuscript.
We followed this suggestion and updated the references to those published in two years.
Please see the references 3-5 in the revised manuscript.
Line 65 and elsewhere in the manuscript: numbers less than ten should be written out in full We followed the suggestion and revised the writing of the numbers.
Line 71 and elsewhere in the manuscript: Avoid abbreviations at the beginning of a sentence We followed the suggestion and revised the sentences. Lines 95-96: Please provide more detailed information for: "To assist epidemiological investigations, food samples (raw chicken, raw ground pork and vegetables)...". As listeriosis is mainly associated with the consumption of ready-to-eat products and is destroyed during cooking, please explain how these results could "assist epidemiological investigations" Raw and undercooked meat and poultry are one of the potential source of bacterial foodborne diseases. Processing and/or preserving the raw meat food products together with cooked or ready-to-eat food products may occur at home, or in some cases in restaurants. Moreover, in some Chinese cuisine styles the raw food products are at high risk of being undercooked. Our findings thus highlight the potential of raw and undercooked meat underlying listeriosis in Taiwan.
Line 102 and elsewhere in the manuscript: insert space between "37" and "{degree sign}C" We followed this suggestion and revised them in the manuscript.
Lines 102-106 and elsewhere in the manuscript: Please avoid long sentences. Rephrase "Then 0.1 ml of the overnight culture was transferred to 10 ml of Listeria enrichment Fraser broth and incubated at 37{degree sign}C, 200 rpm for 18 to 24 hr. The 10 μL of secondary enrichment broth was seeded onto Chromogenic Listeria Agar (CMP, Taiwan) and incubated at 37{degree sign}C for up to 48 h, and the suspected colonies were purified in Brain Heart Infusion Agar (CMP, Taiwan) cultured at 37{degree sign}C for 18 to 24 h.
We rephrased long sentences throughout the manuscript. This part in the method was also revised (L105-110).
The 445 in SRA are those open for public in the Bioproject, and not all the sequences included in this study are publicly available now. Nevertheless, all the 493 SRA sequences included in this study have been uploaded to Bioproject PRJNA493675 and will be made public upon acceptation. The accession numbers of the isolates are provided in the new Table S2. 165-166: "By contrast, all the vegetable products sampled in 2019 (n=50) were negative for L. monocytogenes. " What type of products? These are ready-to-eat mixed salad products sold in supermarkets. We added this information in the revised manuscript (L93-97).
Lines 181: Are the sample products eaten raw?
People in Taiwan do not directly eat these raw meat products. However, processing and/or preserving the raw meat food products together with cooked or ready-to-eat food products may occur at home, or in some cases in restaurants. Moreover, there is a risk that the raw meat products sampled in this study may be undercooked in some Chinese cuisine styles.
Lines 185: or widely distributed CTs?
We agree that these CTs might be widely distributed as we have not investigated the potential contamination of the CTs in food processing and retailing environments as compared to those in soil and water in wild environments. We thus revised the sentence to "suggesting high prevalence of common CTs for several years, which may imply unnoticed long-term outbreaks." (L191-193).
Line 231: in my opinion, we can not claim "epidemiological ... study" We agree that we only have preliminary findings in epidemiology and more complete in genomic surveillance. We followed this comment and revised the sentence to: "Here we described the findings of a genomic nationwide study on clinical and food L. monocytogenes isolates over a 6-year period, prior to and after mandatory notification of human listeriosis cases." (L240-242).
Lines 235-242-"As a result, since July 2021, the Taiwan Food and Drug Administration (TFDA) has extended food monitoring of Listeria from infant and dairy products to refrigerated and ready-to-eat food products (https://consumer.fda.gov.tw). As compared to the regulation before this date where food products were randomly sampled once, an extended sampling plan (5 to 10 sampling times per location according to the guidelines on sampling CAC GL 50-2004) is now in place for food microorganisms. This may help to identify more contamination sources of L. monocytogenes in different food products." Useful information but probably not in this context" Here we discussed how our preliminary findings changed the food sampling policy of the TFDA. The change of the policy may impact L. monocytogenes isolation rates in near future. Nevertheless, we can remove this part if the Editor and Reviewers insist that this is not in the context of the study.
Lines 243-245 "As compared to Europe and North America ...." this should be supported with references from official entities.
The references cited are from official institutions of listeriosis surveillance in U.S.A., Germany and France. We provide now all isolates' detailed information in a supplementary table (Table S2).
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Editor, Microbiology Spectrum Editor comments: The objective needs to be stronger and there needs to be an emphasis that infections being investigated were likely sporadic. In response to reviewer 2 you state: "No cluster infection of L. monocytogenes at a specific place in a short period has been reported in Taiwan. We thus speculated that inappropriate food processing, such as ingestion of undercooked meat, may account for the sporadic listeriosis cases." This is a strong rationale, can this statement or a version of it be incorporated into the text?

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Point-by-point responses
Editor comments: The objective needs to be stronger and there needs to be an emphasis that infections being investigated were likely sporadic. In response to reviewer 2 you state: "No cluster infection of L. monocytogenes at a specific place in a short period has been reported in Taiwan. We thus speculated that inappropriate food processing, such as ingestion of undercooked meat, may account for the sporadic listeriosis cases." This is a strong rationale, can this statement or a version of it be incorporated into the text?
We speculated that sporadic cases might be due to undercooked meat before this study. However, our sequence analyses of the food and clinical isolates identified the major CTs among the isolates, suggesting the potential of clustered listeriosis cases.
We also identified 77 CTs that comprised only 1 isolates within each CT in clinical isolates. We consider these as sporadic cases. We include the information in both results (L187-L188) and discussion (L289-L292). Please see the marked up manuscript for the detail. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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